The proposed research seeks to further our understanding of the three-dimensional binding of the cardiac glycoside, Ouabain, to Sodium- and Potassium Activated Adenosine Triphosphatase (Na+, K+ -ATPase), via photoaffinity labeling studies. The photoaffinity labeling studies will involve the production and assay of a library of mono-diazoacetyl ouabain analogs. The bio-active species will then be crosslinked to the enzyme and the challenging membrane bound protein will be sequenced by tandem mass spectrometry (MS/MS). Our goal is to more thoroughly complete the topographical map of the ouabain binding site.